Terminal differentiation is a process whereby highly specialized cells commit irreversibly to a cellular fate and undergo growth arrest. Recent studies in cardiac muscle have identified two major families of proteins involved in cardiac terminal differentiation, pocket proteins (retinoblastoma gene product (Rb), p107, p130) and the transcriptional coactivators, p300/CBP. One explanation for the dependence of normal differentiation on pocket proteins, is the requirement of some tissue-specific transcription factors on the coexpression of Rb for transcriptional activity. This led us and others to hypothesize that Rb might be the key to terminal differentiation in cardiac myocytes. However, the mechanism underlying this dependence or the factors interacting with Rb are unknown. Therefore, to determine the factors interacting with Rb in cardiac muscle, we utilized the yeast two-hybrid system to identify cardiac Rb- binding proteins. One novel protein, designated p300- and Rb- binding Inhibitor of differentiation-1 (PRI-1) was the predominant Rb-binding clone. PRI-1 is preferentially expressed in cardiac and skeletal muscle in a developmentally regulated manner. Initial studies in skeletal muscle demonstrated that overexpression of PRI-1 inhibited muscle-specific transcription. Repression of skeletal muscle-restricted genes was mediated by a block to transactivation by MyoD, independent of G1 exit, and, surprisingly, was potentiated by a mutation that prevents PRI-1 binding to Rb. Inhibition of MyoD may be explained by PRI-1's ability, like adenoviral protein E1A, to bind p300, an essential MyoD coactivator. Thus, PRI-1 binds both Rb and p300, was inhibited by Rb in differentiated muscle, and was a novel repressor of MyoD function. Studies in cardiac muscle demonstrate that PRI-1 also inhibits cardiac-specific gene expression although the mechanism or potential targets of PRI-1's inhibitory effects are unknown. Thus PRI-1 represents one of the first endogenous, mammalian inhibitors of cardiac differentiation to be identified. This first RO1 proposes to further define the role of PRI-1 in cardiac myocyte differentiation and elucidate its biochemical properties.